Via beta-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen

Using a spleen slice microsuperfusion technique in mice, we have previously characterized the role of norepinephrine (NE) and other neurotransmitters for sympathetic modulation of IL-6 and TNF secretion of splenic macrophages. Since experiments in spleen slices do not reflect the situation of an entire perfused organ, we investigated sympathetic modulation of lipopolysaccharide (LPS)-induced secretion of IL-6 and TNF in perfusion experiments of rat spleen. In an organ bath, perfusion was performed in explanted whole spleens, and catecholamines and cytokines were measured by HPLC and ELISA, respectively. Release of NE depended on stimulation frequency (maximum at 10 Hz). Apart from NE, perfusates also contained significant amounts of dopamine and epinephrine. Furthermore, perfusate epinephrine levels correlated positively with perfusate NE levels (RRank=0.750, p<0.001) but not with plasma epinephrine concentrations. This indicates that epinephrine is a conversion product of sympathetically released NE. Early electrical stimulation of extrasplenic splenic nerves, 20 min after administration of LPS, significantly inhibited TNF secretion. This electrically induced effect was abrogated by simultaneous administration of propranolol (10(-6) M) but it was not influenced by administration of either an alpha1- or alpha2-adrenergic antagonist. Late electrical stimulation of splenic nerves, 2.5 h after administration of LPS, did not change TNF secretion. Interestingly, no influence of early or late sympathetic nerve fiber stimulation on IL-6 secretion was observed. In conclusion, this is the first perfusion study of the entire spleen that demonstrates that early electrical stimulation of sympathetic splenic nerve fibers directly inhibits LPS-induced TNF secretion. This study corroborates the idea that splenic sympathetic nerves are able to inhibit important activators of the innate immune system when stimulation happens very early or even prior to their induction by LPS.     

Either desipramine or TMB-8 suppresses cyanide-induced norepinephrine efflux from in vivo cardiac sympathetic nerves of cats

To investigate the effect of hypoxia on endogenous norepinephrine (NE) release from cardiac sympathetic nerve ending, we administered sodium cyanide (NaCN) for 30 min into the myocardial interstitial space through a dialysis probe and measured dialysate NE levels. During the NaCN perfusion, a marked and concentration-dependent increase in dialysate NE was observed. This cyanide-induced NE response was suppressed by pretreatment with despiramine (a membraneous NE transport inhibitor). Furthermore, the cyanide-induced NE response was suppressed by pretreatment with TMB-8 (intracellular Ca(2+) antagonist) but unaffected by omega-conotoxin GVIA (NE releasing inhibitor). Our data suggest that two (desipramine or TMB-8 suppressive) mechanisms contributed to the amount of NE efflux induced by cyanide in in vivo cardiac sympathetic nerve.     

Norepinephrine suppresses IFN-γ and TNF-α production by murine intestinal intraepithelial lymphocytes via the β₁ adrenoceptor

We examined whether norepinephrine (NE) had direct effects on cytokine production by murine intestinal intraepithelial lymphocytes (IELs), compared with splenocytes. CD3? IELs and CD3? splenocytes expressed α(1B), α(1D), α(2C), β?, β?, and β? adrenoceptors (ARs). NE significantly suppressed IFN-γ and TNF-α production by IELs and splenocytes ex vivo. The suppressive effects of NE in IELs were reversed by β? AR antagonist CGP-20712A, whereas those in splenocytes were reversed by β? AR antagonist ICI118,551. In IELs, β? AR agonist xamoterol mimicked the suppressive effects of NE. These results indicated NE regulates intestinal mucosal immune responses mediated by IELs via β? AR.     

Inhibition of primary murine macrophage cytokine production in vitro following treatment with the K-opioid agonist U50, 488H

Previous work in our laboratory has shown that both μ- and κ-opioid agonists exhibit immunosuppressive activity for antibody responses in vitro. Our earlier work has suggested that both accessory cells and T cells may be altered following treatment with the K-opioid agonist U50, 488H. We intend to further determine the identity of the immune cell population (s) which are affected by opioid treatment, and to determine the nature of the opioid receptor type expressed on these cells. In this study, non-elicited peritoneal macrophages were treated simultaneously with the K-agonist U50.488H and lipopolysaccharide (LPS), and the levels of the cytokines interleukin (IL) -1, IL-6 and tumor necrosis factor (TNF) -α were determined. The results show that U50.488H had a suppressive effect on the production of TNF-α and IL-1 at concentrations as low as 1 nM, while IL-6 was suppressed at concentrations as low as 10 nM. Additional experiments utilizing the opiate antagonist naloxone and the K-selective antagonist norbinaltorphimine (norBNI) were performed in order to further characterize the opioid receptor involved in the cytokine suppression produced by treatment with U50, 488H. Results showed that naloxone was able to partially block U50, 488H suppression while norBNI was able to completely reverse the suppression of IL-6 production. These results suggest that macrophage/monocyte function is significantly modulated following activation of the K-opioid receptor.     

Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.     

Activation of the hypothalamic-pituitary-adrenal axis differentially affects the anti-mycobacterial activity of macrophages from BCG-resistant and susceptible mice

The effect of hypothalamic-pituitary-adrenal (HPA) axis activation and exogenous glucocorticoids on the ability of splenic macrophages to control the growth of Mycobacterium avium was evaluated. We found that activation of the HPA axis by restraint stress or the addition of corticosterone increased the susceptibility of macrophages from mice that are innately susceptible to the in vivo growth of M. avium. In contrast, the ability of macrophages from innately resistant, congenic mice to control the growth of M. avium was not affected by HPA activation or the addition of corticosterone. The effect of restraint and of corticosterone on macrophage function was abrogated by either treating mice with the glucocorticoid receptor antagonist RU486 or the addition of the drug to cultures of macrophages. Activation of the HPA axis as well as the addition of corticosterone to cultures of macrophages resulted in a suppression of the production of tumor necrosis factor (TNF)-α and of reactive nitrogen intermediates by macrophages from both strains of mice. The lack of effect of HPA activation and of corticosterone on the mycobacterial resistance of macrophages from BCG-resistant mice, while at the same time suppressing the production of reactive nitrogen intermediates, appears to rule out a role for this antimicrobial pathway in innate resistance to mycobacterial growth.     

A longitudinal study of age-related loss of noradrenergic nerves and lymphoid cells in the rat spleen

A longitudinal study of sympathetic noradrenergic (NA) innervation of the spleen was carried out in 3-, 8-, 12-, 17-, 21-, and 27-month-old Fischer 344 (F344) rats using (i) fluorescence histochemistry for localization of norepinephrine (NE); (ii) immunocytochemistry (ICC) for localization of tyrosine hydroxylase (TH)-positive nerve fibers alone, and in combination with specific markers for T and B lymphocytes (OX19 and anti-mu respectively), and macrophages (ED3); and (iii) high-performance liquid chromatography with electrochemical detection for quantitation of NE. Fluorescence histochemistry revealed extensive loss of NA nerve fibers in all compartments of the spleen in 21- and 27-month-old rats. With single-label ICC, a decline in TH+ nerve fibers in all compartments of the spleen was observed by 17 months of age and became more severe with advancing age; these findings suggest that both the rate-limiting enzyme and the transmitter itself (NE) are depleted from sympathetic nerves in aged rat spleen. Double-label ICC demonstrated the loss of TH+ nerve fibers in spleen from 17-, 21-, and 27-month-old rats, and a parallel loss of OX19+ T lymphocytes and ED3+ macrophages in these cellular compartments. Neurochemical measurement of NE demonstrated a decline in NE per wet weight at 27 months of age. The age-related decline in NA innervation of spleen and in the density of specific populations of cells of the immune system (T lymphocytes and antigen-presenting ED3+ macrophages), that follow remarkably similar time courses, supports functional evidence for dynamic interactions between the immune system and NA sympathetic nerves in the spleen, and further suggests a causal relationship between these age-related phenomena, i.e., that age-related immunosenescence promotes sympathetic denervation of the spleen which further compromises immune function. This hypothesis, however, requires further testing.     

The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice

The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.     

The vagus nerve modulates CD4+ T cell activity

The vagus nerve has a counter-inflammatory role in a number of model systems. While the majority of these anti-inflammatory effects have been ascribed to the activation of nicotinic receptors on macrophages, little is known about the role of the vagus in modulating the activity of other cells involved in inflammatory responses. Here, we demonstrate that following subdiaphragmatic vagotomy of mice CD4⁺ T cells from the spleen proliferated at a higher rate and produced more pro-inflammatory cytokines, including TNF and IFN-γ, upon in vitro stimulation. Cell responses were restored to control levels following the administration of nicotine and the treatment of non-vagotomized animals with a nicotinic receptor antagonist could mimic the effect of vagotomy. Our results suggest that vagal input constitutively down-regulates T cell function through action at nicotinic receptors and the role of the vagus in regulating immune responses is more extensive than previously demonstrated.     

Role of sympathetic nervous system in the entrainment of circadian natural-killer cell function

Previous research in our laboratory has demonstrated robust circadian variations of cytokines and cytolytic factors in enriched NK cells from rat spleen, strongly suggesting these functions may be subject to circadian regulation. The SCN mediates timing information to peripheral tissues by both humoral and neural inputs. In particular, noradrenergic (NE) sympathetic nervous system (SNS) terminals innervate the spleen tissue communicating information between central and peripheral systems. However, whether these immune factors are subject to timing information conveyed through neural NE innervation to the spleen remained unknown. Indeed, we were able to characterize a circadian rhythm of NE content in the spleen, supporting the role of the SNS as a conveyor of timing information to splenocytes. By chemically producing a local splenic sympathectomy through guanethidine treatment, the splenic NE rhythm was abolished or shifted as indicated by a blunting of the expected peak at ZT7. Consequently, the daily variations of cytokine, TNF-α, and cytolytic factors, granzyme-B and perforin, in NK cells and splenocytes were altered. Only time-dependent mRNA expression of IFN-γ was altered in splenocytes, but not protein levels in NK cells, suggesting non-neural entrainment cues may be necessary to regulate specific immune factors. In addition, the rhythms of clock genes and proteins, Bmal1 and Per2, in these tissues also displayed significantly altered daily variations. Collectively, these results demonstrate rhythmic NE input to the spleen acts as an entrainment cue to modulate the molecular clock in NK cells and other spleen cells possibly playing a role in regulating the cytokine and cytolytic function of these cells.     

Calcitonin gene-related peptide receptors in pig spleen and the involvement of the CGRP(1)receptor in the splenocyte function

Calcitonin gene-related peptide (CGRP)-positive nerve fibers have been found in the trabecula and parenchyme area of pig spleen. Receptor studies have demonstrated that the CGRP binding site in pig spleen membranes has an average K(d)2.24 +/- 0.48 nM and B(max)78 +/- 4.09 fmol/mg of protein. In the K(d)range demonstrated in the binding studies, the dose-dependent suppressive effect of CGRP on spleen T lymphocyte proliferation was found with the maximal effect in 10(-9)M concentration. The same effect, but in a different concentration, was found on peripheral blood T lymphocytes with the maximum in 10(-6)M concentration. Contrary to the results obtained through the simultaneous presence of CGRP and mitogen, preincubation with CGRP led to a stimulation of peripheral blood lymphocytes in response to ConA and had no effect on spleen T lymphocytes. These results illustrate the difference in CGRP effect between lymphocytes of different origins. Using CGRP(1)receptor antagonist CGRP(8-37), we established that the CGRP suppressive effect on spleen T lymphocyte proliferation is CGRP(1)-receptor mediated.     

Amitriptyline administration transforms tumor necrosis factor-alpha regulation of norepinephrine release in the brain

The present study demonstrates that the mixed action antidepressant drug amitriptyline enhances norepinephrine (NE) release by transforming the nature of the response of neurons to both tumor necrosis factor-alpha (TNF) as well as to an alpha(2)-adrenergic agonist in an area of the central nervous system (CNS) rich in adrenergic neurons. Administration of the antidepressant drug amitriptyline for 1 day or 14 days to rats significantly increases TNF bioactivity in total homogenates of the locus coeruleus (LC) and the hippocampus as assessed by the WEHI-13VAR bioassay. Superfusion and electrical field stimulation of rat hippocampal brain slices were used to study the regulation of NE release. Exposure to TNF, as well as activation of the alpha(2)-adrenergic autoreceptor inhibits stimulation-evoked norepinephrine (NE) release from adrenergic neurons of the CNS from na?ve rats. Superfusion of hippocampal slices isolated from rats chronically (14 days) administered amitriptyline demonstrates that TNF inhibition of NE release is transformed, such that TNF facilitates NE release, dependent upon alpha(2)-adrenergic activation. Furthermore, chronic administration of amitriptyline increases stimulation-evoked NE release and decreases alpha(2)-adrenergic autoreceptor inhibition of NE release, an effect not observed with acute drug administration. These data support the hypothesis that chronic antidepressant drug administration, through regulation of TNF expression, transforms alpha(2)-adrenergic receptors such that they function to facilitate NE release, suggesting a mechanism of action of antidepressant drugs.     

Acute stress suppresses pro-inflammatory cytokines TNF-alpha and IL-1 beta independent of a catecholamine-driven increase in IL-10 production

Interleukin (IL)-10 is an anti-inflammatory cytokine that can down-regulate various aspects of the immune response. In this study we demonstrate that exposure to a psychophysiological stressor (swim stress) increases IL-10 production in female rats in response to an in vivo challenge with bacterial lipopolysaccharide (LPS). This increase in LPS-induced IL-10 was associated with suppression of the pro-inflammatory cytokines IL-1beta and TNF-alpha, indicating that overall, swim stress promotes an immunosuppressive cytokine phenotype. Despite the well-documented ability of IL-10 to suppress pro-inflammatory cytokine production, neutralisation of IL-10 failed to block the stress-induced suppression of IL-1beta and TNF-alpha. These data indicate that the suppressive effect of swim stress on these pro-inflammatory cytokines occurs independently of increased IL-10 production. To determine if swim stress-induced immunosuppression was mediated by increased sympathetic nervous system activity, and subsequent beta-adrenoceptor activation, we assessed the ability of the beta-adrenoceptor antagonist nadolol to block stressor-induced changes in cytokine production. Whilst pre-treatment with nadolol completely blocked the stress-induced increase in IL-10, it failed to alter the suppression of TNF-alpha or IL-1beta. Similarly, pre-treatment with the glucocorticoid receptor antagonist mifepristone also failed to attenuate the suppressive effect of swim stress on IL-1beta and TNF-alpha production. These data indicate that neither increased glucocorticoid secretion, nor catecholamine-induced beta-adrenoceptor activation, mediates the suppressive effect of swim stress on pro-inflammatory cytokine production. Taken together, these data demonstrate a role for beta-adrenoceptor activation in the ability of acute swim stress to increase LPS-induced IL-10 production, and also highlight a mechanistic dissociation between the ability of swim stress to increase IL-10 and suppress pro-inflammatory cytokine production.     

VEGF as a modulator of the innate immune response in glioblastoma

VEGF is an important factor in tumor vascularization and used as target for anti‐angiogenic treatment strategies in glioma. In this study, we demonstrate for the first time that VEGF is a modulator of the innate immune response with suppressive effects on the immunologic and pro‐angiogenic function of microglia/macrophages in a glioblastoma rodent model. High level of VEGF led to threefold enlarged tumor volumes and a pronounced remodeling of the vascular structure along with a reduced infiltration of microglia/macrophages by approximately 50%. Remaining microglia/macrophages showed an enhanced rate of apoptosis as well as significant downregulation of the VEGF‐receptor, VEGFR2, and others such as CXCR4. Consequently, we determined a substantially impaired migration of these microglia/macrophages to VEGF and SDF1α in vitro. Furthermore, we observed an increased presentation of the surface molecules MHCI and MHCII on microglia/macrophages from VEGF‐overexpressing gliomas that are essential for activation of the adaptive immune system. In contrast, the expression of pro‐inflammatory and suppressive cytokines, associated with the innate immune response, were mainly downregulated. Remarkably, the abundance of VEGF provoked less accumulation of microglia/macrophages within the perivascular niche and concomitantly reduced the release of pro‐angiogenic factors, like VEGF, suggesting a possible regulatory feedback mechanism. Thus, the quantity of VEGF in the glioma microenvironment seems to be crucial for the participation of microglia/macrophages on tumor progression and should be considered for developing novel therapeutic approaches.     

In vitro superfusion method for the investigation of nerve-immune cell interaction in murine spleen

Immune defence mechanisms can be modulated by brain function. To study such interactions, an in vitro method was developed to examine the release of cytokines and norepinephrine (NE) after electrical stimulation. Slices of mouse spleen were placed in chambers with a volume of 80 μl and superfused with culture medium. To characterize electrically evoked NE release and cell viability a suitable stimulation protocol was developed using of [³H]NE. As parameter for immune function, modulation of interleukin-6 (IL-6) release by the spleen cells brought about by electrical stimulation was investigated. Splenic [³H]NE overflow was calcium-dependent, tetrodotoxin-sensitive and elicited by 54 mM potassium. Electrically evoked [³H]NE release at 22 h was about 80% of the release at 5.3 h. Electrical stimulation substantially reduced IL-6 secretion at 6 h (control: 143.4 ± 14.3 vs. electrical: 71.3 ± 7.9 pg/ml/10⁶ leukocytes, P = 0.0001). This effect was inhibited in a concentration-dependent manner by the β-adrenergic antagonist propranolol (P = 0.0298, EC₅₀approx. 10⁻⁷M). In conclusion, this new technique allows long-term investigation of cell function in slices of murine spleen. In addition, these are the first in vitro data indicating the presence of a functional neuroimmunological link in murine lymphoid tissue.     

Hypophysectomy disturbs the noradrenergic feeding system of the paraventricular nucleus

Injection of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN) of satiated rats is known to stimulate eating behavior. In addition, drinking behavior is potentiated just prior to the onset of eating, followed by a strong inhibition of water intake. To understand the relationship between these PVN noradrenergic phenomena and endocrine processes associated with the PVN, chronically hypophysectomized animals were tested for their behavioral responsiveness to PVN NE injection. Pituitary ablation was found to abolish the NE-elicited eating response and the NE drinking suppressive effect. However, hypophysectomy had no impact on the NE-elicited preprandial drinking response, nor did it affect drinking produced by carbachol, angiotensin, and histamine, or the feeding and drinking responses induced by insulin. These results demonstrate that hypophysectomy disturbs PVN noradrenergic mechanisms in a behaviorally and pharmacologically specific specific manner.     

Cytokine-induced change in hypothalamic norepinephrine turnover: involvement of corticotropin-releasing hormone and prostaglandins

Changes in norepinephrine (NE) turnover in restricted brain regions were examined in rats after administration of the major mediators of the acute phase response, interleukin-1β (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF). An increase in NE turnover was observed after intraperitoneal injection of IL-1 (1 μg/rat) in the whole hypothalamus and several specific hypothalamic nuclei, but not in the medulla oblongata and cerebral cortex. The stimulatory effect of IL-1 was mimicked by an intracerebroventricular injection of much lower doses of IL-1 (10–100 ng/rat). This IL-1-induced increase in hypothalamic NE turnover was blocked by the pretreatment with either indomethacin (cyclooxygenase inhibitor) or anti-corticotropin releasing hormone (CRH) antibody but not by naloxone. Intracerebroventricular injection of CRH increased NE turnover not only in the hypothalamus but also in the medulla oblongata and cerebral cortex. However, prostaglandin (PG) E₂ and PGF_2α did not show such effect. It was therefore suggested that IL-1 activates noradrenergic neurons projecting to the hypothalamus by its direct action to the brain, and that CRH and eicosanoid-cyclooxygenase product(s) within the brain are involved in this process. In contrast, neither IL-6 nor TNF influenced brain NE turnover regardless of whether they were given intraperitoneally or intracerebroventricularly. Thus, although IL-6 and TNF, as well as IL-1, show common central effects such as fever and pituitary-adrenal activation, these effects may be independent of the activation of NE metabolism in the hypothalamus.     

Effect of tumor necrosis factor-alpha on the reciprocal G-protein-induced regulation of norepinephrine release by the alpha2-adrenergic receptor

Alpha2-adrenergic receptors control norepinephrine (NE) release and tumor necrosis factor-alpha (TNF) production from neurons. TNF regulates NE release, depending on alpha2-adrenergic receptor functioning. The relationship between TNF production in the brain and alpha2-adrenergic receptor activation could have profound control over NE release. TNF and alpha2-adrenergic regulation of NE release was investigated in rat hippocampal slices incubated with pertussis toxin (PTX). The alpha2-adrenergic receptor couples to Galpha(i/o)-proteins to inhibit NE release; however, in slices preexposed to PTX, alpha2-adrenergic receptor activation facilitates NE release. TNF exposure subsequent to PTX restores alpha2-adrenergic inhibition of NE release. PTX exposure of hippocampal slices prevents agonist-induced increases in Galpha(i/o) labeling with a GTP analog; after subsequent TNF exposure, agonist-induced increases in Galpha(i/o) labeling are restored. TNF regulation of NE release transforms from inhibition to facilitation depending on alpha2-adrenergic receptor activation following PTX exposure. Therefore, TNF directs the coupling of the alpha2-adrenergic receptor, ultimately affecting NE release.     

Electrophysiological characterization of adrenoceptors in the rat dorsal hippocampus. II. Receptors mediating the effect of synaptically released norepinephrine

The present studies were undertaken to determine the nature of the receptors mediating the effects of endogenous norepinephrine (NE) released by stimulation of the locus coeruleus (LC) on the firing activity of dorsal hippocampus pyramidal neurons in the rat. Unitary activity of CA3 pyramidal neurons was recorded extracellularly. In most neurons, the LC stimulation produced a period of suppression, followed by a period of activation. The suppression was selectively blocked by prazosin, an alpha 1-adrenoceptor antagonist, whereas the activation was selectively blocked by propranolol, a beta-adrenoceptor antagonist. Idazoxan, an alpha 2-adrenoceptor antagonist, increased the period of suppression without affecting the period of activation. The effectiveness of microiontophoretic applications of NE on the same neurons was reduced by idazoxan, but was modified neither by propranolol nor prazosin. Lesion of the central noradrenergic system by intracerebroventricular 6-hydroxydopamine markedly decreased the NE content in the hippocampus in all rats but the effectiveness of the LC stimulation was reduced only in rats with a depletion greater than 90%. These results demonstrate that the suppressant effect of endogenous NE released by LC stimulation on hippocampus pyramidal neurons is mediated by an alpha 1-adrenoceptor and suggest that its late excitatory effect might involve beta-adrenoceptors. Since the effect of microiontophoretically applied NE on the same neurons is mediated by alpha 2-adrenoceptors, these data provide evidence that, in the rat hippocampus, postsynaptic alpha 1-adrenoceptors are intrasynaptic, whereas postsynaptic alpha 2-adrenoceptors are extrasynaptic.     

Temporal effects of left versus right middle cerebral artery occlusion on spleen lymphocyte subsets and mitogenic response in Wistar rats

The left and right neocortex of the brain has been shown to exert asymmetrical effects on the immune system. In the present study, we used a middle cerebral artery (MCA) occlusion model in Wistar rats to analyze the influence of unilateral CNS ischemia on spleen cell number and function. The occlusion time was 1 h, followed by reperfusion with survival for 0, 2, 7, 14, and 28 days. Changes in plasma norepinephrine levels were used as an index of peripheral sympathetic activity. Results showed that the total number of spleen cells significantly decreased after 2-28 days of survival in animals with cerebral ischemia compared to sham-operated controls. There was no change in the percentage of CD5(+)-CD4(+) T cells, MHC class II(+) cells, or ED1(+) macrophages. However, the percentage of CD5(+)-CD8(+) T cells decreased at 2 days, resulting in an increased CD4/CD8 ratio, and both parameters returned to control levels after 7 days. Mitogen-induced T and B lymphocyte proliferation increased after 0-28 days post-ischemia independently of the mitogen used. There was no difference in immune response or norepinephrine levels between left and right MCA occlusions. These results are consistent with the notion that cerebral ischemia induces mobilization of certain immune cells from the periphery to the brain, where they may contribute to the local inflammatory response. Additionally, the data indicate that cerebral ischemia is followed by a systemic activation of T and B lymphocytes. Absence of asymmetric effects of left versus right stroke, and failure to demonstrate any suppressive effects of left-sided lesions on lymphocyte proliferation, probably reflects the fact that these large cerebral ischemic lesions affect both cortical and subcortical areas.